Akasaka S, Yamamoto K
Division of Industrial Health, Osaka Prefectural Institute of Public Health, Japan.
Mutat Res. 1994 Sep;315(2):105-12. doi: 10.1016/0921-8777(94)90011-6.
In vitro incubation of rat microsomal lipids with NADPH and Fe3+ in the presence of cytochrome P450 reductase produces lesions in double-stranded pZ189 plasmid DNA, the mutagenic potential of which was analyzed after transfection into Escherichia coli host cells that had been induced for SOS functions by ultraviolet irradiation. The extent of lipid peroxidation, when monitored by the formation of thiobarbituric acid reaction substances, was increased with increased addition of lipids in the reaction mixture. Mutagenesis was determined with the forward mutation assay using the supF gene of pZ189 as a target. When treated pZ189 DNA was used to transfect host cells, a seven-fold increase in mutation frequency for SOS uninduced hosts and a 12-fold increase in mutation frequency for SOS induced hosts was observed at 50% survival compared to that observed with untreated DNA. Sequence analysis shows that incubation of pZ189 DNA in the lipid peroxidation reaction mixture results mostly in single base substitutions, the most frequent base change being G:C-->C:G transversion, followed by G:C-->T:A transversion. The fact that, in the SOS induced hosts, the spectrum obtained by lipid peroxidation is similar to that of hydrogen peroxide suggests the possible involvement for mutagenesis of superoxide produced during lipid peroxidation, but not lipid peroxidation end products such as aldehyde or alkane. Treatment of pZ189 DNA with increasing extents of lipid peroxidation did not yield increasing formation of 8-hydroxyguanine. The results suggest that the origins of G:C-->C:G and G:C-->T:A transversions may be (an) as yet unidentified lesion(s) generated by lipid peroxidation.
在细胞色素P450还原酶存在的情况下，将大鼠微粒体脂质与NADPH和Fe3+进行体外孵育，会使双链pZ189质粒DNA产生损伤。在将其转染到经紫外线照射诱导产生SOS功能的大肠杆菌宿主细胞后，分析了其诱变潜力。当通过硫代巴比妥酸反应物质的形成来监测脂质过氧化程度时，反应混合物中脂质添加量增加，脂质过氧化程度也随之增加。使用pZ189的supF基因作为靶点，通过正向突变试验来确定诱变作用。当用处理过的pZ189 DNA转染宿主细胞时，与未处理的DNA相比，在50%存活率时，SOS未诱导宿主的突变频率增加了7倍，SOS诱导宿主的突变频率增加了12倍。序列分析表明，pZ189 DNA在脂质过氧化反应混合物中的孵育主要导致单碱基取代，最常见的碱基变化是G:C→C:G颠换，其次是G:C→T:A颠换。在SOS诱导宿主中，脂质过氧化产生的光谱与过氧化氢产生的光谱相似，这一事实表明脂质过氧化过程中产生的超氧化物可能参与诱变作用，而不是醛或烷烃等脂质过氧化终产物。用脂质过氧化程度不断增加的处理pZ189 DNA，并未导致8-羟基鸟嘌呤的形成增加。结果表明，G:C→C:G和G:C→T:A颠换的起源可能是脂质过氧化产生的一种（或多种）尚未确定的损伤。
